PROJECT SUMMARY/ABSTRACT Hypertrophic cardiomyopathy (HCM) patients are at risk for sudden cardiac death and progressive heart failure (HF), and there are no effective therapeutics, due in part to our limited genetic understanding of HCM pathogenesis. There are critical gaps in our current knowledge of the molecular mechanisms that link specific mutations in the beta myosin heavy chain gene (MYH7) to pathological thickening of the heart muscle that is associated with HCM. Our long-term goals are to utilize genomic tools combined with 3-dimensional cardiac microtissues derived from human induced pluripotent stem cells to interrogate mechanisms of HCM secondary to specific MYH7 variants, and to utilize these insights to identify new disease biomarkers and therapeutic targets for specific HCM patients. Our previous studies identified that two HCM-associated MYH7 variants, arginine 403 to glutamine and valine 606 to methionine that are located in the actin-binding domain of beta myosin heavy chain protein (MHC- ?), generate increased microtissue contraction force with associated abnormalities in contraction kinetics. Studies by others have indicated that MYH7 variants located in distinct structural domains of MHC-? cause distinct phenotypes. These results lead to our central hypothesis that HCM is a heterogeneous disorder, in which patient symptoms and therapeutic responses are dependent on the location of the causative MYH7 variant(s) within the gene and on cell-type specific transcriptional and epigenetic programs, which initiate from abnormalities in contractile function. Guided by our comprehensive preliminary data, we propose to pursue three Specific Aims to determine multi-scale insights into HCM pathogenesis: (1) to characterize functional consequences of MYH7 variants localized to the actin-binding, ATP-binding and converter domains of MHC- ?, (2) to identify cell type-specific transcriptional and epigenetic mechanisms of HCM in microtissues using paired single-cell RNA-seq and ATAC-seq and (3) to interrogate the function of C1ORF105, a nuclear-encoded mitochondrial protein that is associated with HCM and is specifically expressed in human cardiomyocytes. In summary, the execution of these aims will provide a more precise understanding of the functional role of MYH7 variant localization, generate novel cell-type specific and molecular mechanisms of HCM and identify critical molecular linkages between sarcomere and mitochondrial function that will broadly impact the field of HCM and heart failure.